MSF Proteomics and Protein Characterization

FREQUENTLY ASKED QUESTIONS

SAMPLE PREPARATION

What types of samples can I send?

  • We accept samples of all types, including tissue, cells, plasma/serum, other bodily fluids (e.g. urine, lavage, etc.), synthetic compounds, environmental samples, and more.
  • Samples we do NOT accept include:
    • Radioactive-labeled samples
    • Samples containing pathogenic or harmful substances that have not been deactivated first using acceptable inactivation protocols**
**Please contact us for acceptable methods to inactivate samples containing pathogenic or harmful substances.

How much sample should I send?

  • The following are the preferred amounts of sample we will need to analyze:
    • Label-free quantitation proteomics: 5-100 μg protein/sample
    • Affinity purified MS proteomics: 5-50 μg protein/sample
    • Tissue for proteomics: 5-30 mg
    • Cells for proteomics: 1-2e6 PBS-washed pellets or 1-2e6 cell suspensions
    • Phospho proteomics: 0.5-1 mg protein
    • Ubiquitin proteomics: 10 mg protein
    • For other sample types, please contact us directly for more information

  • When sending samples, be sure to indicate the sample weight, cell number, or concentration and volume on your Sample Submission Form.
  • For protein characterization, we perform an intact protein measurement and a peptide map. For most projects:
    • We prefer to have samples at 50 pmol/μL (or 5 mg/mL mAb) in 40 μL volume for intact characterization.
    • For peptide mapping with sequence characterization we would like 10 pmol/μL (or 1 mg/mL mAb) in 40 μL volume

How many samples can I send?

  • You may send as few as one to as many as several hundred. However, for optimal results we recommend 5 bioreplicates from the different experimental groups (e.g. treated vs. untreated, WT vs KO, etc.), but a minimum of 3 samples per sample group are needed for proper statistical analyses

How should I send my samples?

  • Frozen samples should be shipped to MSF on dry ice using overnight shipping. Samples containing IP beads in wash buffer should be shipped on ice packs using overnight shipping. We recommend shipping by Tuesday morning to avoid complications with delivery. Please ship to:

UTMB Mass Spectrometry Facility, L04565
UTMB Central Receiving
14th and Strand
Galveston, TX 77555-1027
(Lab): 409-772-6338
Office: 409-772-3579

What other information should I send?

  • Please include a Sample Submission Form with your shipped samples. (Note that the online submission form is the preferred method)
    • For UTMB on-campus samples, an ISC# must be provided before samples will be processed
    • For off-campus samples, credit card information must be provided to Nathan Camardo (npcamard@utmb.edu) before samples will be processed

  • On the Sample Submission Form, please include the following:
    • All species the samples are from, for example if you ware expressing a human protein in E. coli, we would require you to add both human and E. coli to the form.
    • Sample names/IDs and sample groups

  • A brief description of the project is also welcome as it will help us look into the background literature for a better understanding of how best to analyze your samples. Providing examples from the literature of the type of analysis you desire is greatly appreciated.

How much does it cost to analyze samples?

  • This will depend on how many samples you are submitting, what assay(s) you would like to be done, and how the data are analyzed afterwards. Please contact Prof. Russell (bill.russell@utmb.edu) for a quote on specific costs.

LC-MS ANALYSIS

How will my samples be analyzed?

  • Samples are digested, typically with trypsin, and cleaned up by one of the MSF staff members and then run by LCMS either in data dependent or independent mode.
  • Our proteomics analyses are performed on either a ThermoScientific Orbitrap Eclipse or Fusion coupled to nanoRSCL 3000 HPLC or EvoSep One.
  • Intact proteins are analyzed using a ThermoScientific Vanquish coupled to either the Eclipse or Fusion.
  • Custom targeted peptide assays can be created in which we will optimize ionization and LC gradient conditions using verified stable isotope peptide standards to develop or add to existing methods. Standards are typically supplied by the client.
  • Once samples are injected and the data is acquired, we identify and quantify the data, and perform statistical analyses using a variety of software (Proteome Discoverer, Frag-pipe, PEAKS, Biopharma Finder, or Skyline).

What is the difference between absolute and relative quantitation? Which is best for my samples?

  • Absolute quantitation uses stable isotope labeled homologues of the peptides to be measured as internal standards.** Usually these standards are spiked into samples prior to extraction. Limits of detection and quantitation (LOD and LOQ) are determined with the linearity of response to determine accurate quantitation of absolute values. This type of quantitation provides quantitative values, such as “nmol/mg”, “μM”, or other units.
  • Relative quantitation uses surrogate internal standards that are chemically similar to a class of compounds but may not be exactly the same. One or a few surrogate internal standards are included to account for many different types of peptides, and they may or may not be isotopically-labeled. This type of quantitation is useful for determining “up/down” changes between sample types, such as wildtype vs. mutant, or treated vs. control. This type of quantitation may be desired if absolute quantitation is unnecessary to the research goals. Values are represented as “Relative amount” or “Fold difference” or “Peak area ratio” or other arbitrary units.
  • The choice of which quantitation to choose will largely depend on whether absolute, quantitative values are needed or desired, otherwise our analyses will generally default to relative quantitation.
**The use of isotopically-labeled internal standards may increase the cost of analysis depending on the assay requested.

DATA PROCESSING

How will the proteomics data be returned?

  • We use either Proteome Discoverer or FragPipe (MSFragger for identification and ionQuant for DDA quantitation and DiaNN for DIA quantitation) for data analysis depending upon the request. Generally, we provide either text files for visualization using a web interface or a PowerPoint that has an overview of the methods used and the key findings of the analysis, which may include bar charts, scatter plots, heatmaps, or other statistical tests and figures. We also provide an Excel document that contains all the processed data and the LC-MS peak integration values before processing. Processing of the data will usually involve normalization, averaging and standard deviations of replicates within sample groups, and statistical analyses where appropriate.

Is the raw data available? Will it be uploaded to a publicly available database?

  • We can provide the raw data files upon request. However, we cannot provide the software** used to analyze the data due to licensing restrictions on commercial software or other open-source software that we use is already available elsewhere.
  • We do not upload raw data to public databases unless requested.
**We can provide guidance and support if you decide to analyze raw data yourself using your own copy(ies) of data analysis software.

How long will it take for samples to be analyzed?

  • This is highly dependent on the number of samples submitted, how many samples from other labs are currently being analyzed in the core facility or waiting to be analyzed, whether method development is required, and the complexity of the analysis involved. However, in general we can analyze samples and return data within 2-3 weeks of receiving samples.

What if my data doesn’t look right?

  • Contact us immediately if you believe there are discrepancies in your data. We hold on to sample extracts in -80°C freezer storage if they require re-analysis, and we can always reanalyze already acquired data if there are issues there.

PUBLISHING DATA COLLECTED BY MSF

Do I need to list an MSF member as a coauthor?

  • While this is not a requirement, if you believe a staff member of MSF provided in-depth analysis, experimental design, or otherwise contributed to the intellectual merits of the project, then we would greatly appreciate being included as a coauthor.

How should I describe the methods under “Materials and Methods” sections of a paper?

  • The reports we provide following an analysis will have an abbreviated description of the methods used to analyze your samples. If you require a more detailed description, we can provide this as needed.

Do I need to acknowledge MSF?

  • YES, please acknowledge MSF within the acknowledgments section of any papers that use data produced by MSF.
  • MSF is also supported in part by Cancer Prevention Research Institute of Texas (CPRIT) grant numbers RP190682 & RP250644. Please acknowledge this funding in your publication.